EXPERT REVIEW OF RESPIRATORY MEDICINE

https://doi.org/10.1080/17476348.2021.1917389

REVIEW

False-positive and false-negative COVID-19 cases: respiratory prevention and

management strategies, vaccination, and further perspectives
Dimitra S. Mouliou and Konstantinos I. Gourgoulianis
Department of Respiratory Medicine, University of Thessaly, Larissa, Greece

ABSTRACT ARTICLE HISTORY

Introduction: A novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was reported via Received 18 February 2021
nucleic acid identification in December, 2019. Accuracy of SARS-CoV-2 diagnostic assays has emerged Accepted 12 April 2021
as a major barrier to COVID-19 diagnosis, particularly in cases requiring urgent or emergent treatment. KEYWORDS
Areas covered: In this review, we explore the major reasons for false-positive and false-negative SARS- COVID-19; false-negative;
CoV-2 test results. How clinical characteristics, specific respiratory comorbidities and SARS-CoV-2 false-positive; management;
vaccination impact on existing diagnostic assays are highlighted. Different COVID-19 management respiratory; vaccination
algorithms based on each test and limitations are thoroughly presented.
Expert opinion: The diagnostic accuracy and the capacity of every available assay, which need to be
interpreted in the light of the background incidence of SARS-CoV-2 infection in the communities in
which they are used, are essential in order to minimize the number of falsely tested cases. Automated
testing platforms may enhance diagnostic accuracy by minimizing the potential for human error in
assays’ performance. Prior immunization against SARS-CoV-2 impairs the utility of serologic testing of
suspected COVID-19 cases. Future avenues of research to evaluate lung tissue innate immune responses
hold promise as a target for research to optimize SARS-CoV-2 and future infections’ testing accuracy.

1. Introduction The current massive use of RDTs by inexperienced indivi­

duals, and poor rRT-PCR laboratory procedures, increase the
Three highly pathogenic coronaviruses have impacted sub­
risk of a false-positive test result. Consequently, challenges
stantially on human populations since the beginning of the
arise in hospitalizations and treatments when needed, epide­
21st century. In December 2019, a novel coronavirus
miological studies may overestimate the extent of disease,
(SARS-CoV-2) was reported from a cluster of pneumonia
financial and business losses emerge from forced isolation in
cases in Wuhan, China [1]. As defined by World Health
response to false-positive tests, and adverse psychological and
Organization (WHO), a confirmed case is detected from
societal effects arise through lockdown policies which are
nucleic acid amplification tests (NAAT) for SARS-CoV-2,
designed to limit transmission of SARS-CoV-2 in commu­
such as real-time reverse transcription polymerase chain
nities [6].
reaction (rRT-PCR), that is worldwide preferred [2].
Reversely, people infected with SARS-CoV-2 but tested
Nowadays, immunometric assays (IMAs) are performed for
negative, remain unaware of their infection status, and may
detecting the immune response to COVID-19, while several
develop a false sense of security based on their test results,
test manufacturers have launched various rapid diagnostic
and pose a risk for onward transmission of the virus. This
tests (RDTs), to facilitate SARS-CoV-2 detection at point-of-
would give rise to a situation that perpetuates local epidemics,
care.
placing people at high risk for a severe COVID-19.
It is widely accepted that tests are not completely fool­
False-positive tests have generally attracted more attention
proof, and, thus no single ‘gold standard’ assay exists. One
than false-negative tests, but both are important in successful
or more negative tests do not rule out the possibility of
management of local disease epidemics. The accuracy of avail­
SARS-CoV-2 infection [2–4]. Retrospectively, test positivity
able tests must be optimized, particularly within the context of
does not always show an infection existing in reality. The
increasing access to SARS-CoV-2 vaccination which further
false-negative rRT-PCR test probability for SARS-CoV-2
impacts on interpretation of serologic tests for COVID-19.
depends on various sampling and technical factors,
whereas the chances of obtaining a true positive result
decreases over time, and with decreasing viral titers in 2. False COVID-19 cases
clinical specimens [2–5]. Principally, false-positive tests
2.1. False-positive test results
refer to a wrong indication for a particular infection to
be present, while false-negative tests pertain to patients Initially, rapid diagnosis was recommended by WHO mainly in
labeled as being ‘uninfected’, despite being infected. research; however, low-cost technologies with a high degree

CONTACT Dimitra S. Mouliou [email protected] Department of Respiratory Medicine, University of Thessaly, Biopolis, Larissa 41110, Greece
© 2021 Informa UK Limited, trading as Taylor & Francis Group
2 D. S. MOULIOU AND K. I. GOURGOULIANIS

different ways [18,19]. Certain Ig-RDTs detect SARS-CoV-2 specific

Article highlights antibodies, antibodies to other viruses, antinuclear antibodies and
● False-positive COVID-19 cases occur in erroneous testing and cross-
other autoantibodies [15,20]. Wang et al reported false-positive
reactions, and place patients at risk through cohorting with other SARS-CoV-2 antibody tests in the face of rheumatoid factor, while
COVID-19 cases. Tan et al described possible cross-reactivity with HIV [21,22]. LFIAs
● False-negative COVID-19 cases occur through sample deficiency, con­
current respiratory infection, or test inhibitors, and place healthcare
may also be affected by the presence of heterophilic antibodies,
workers, other patients, and the general public at risk for infection such as human anti-mouse antibodies (HAMA), which have also
from an undiagnosed source case. been described as giving rise to false-positive results [23,24].
● SARS-CoV-2 vaccination produces an antibody response, which ren­
ders serologic testing for COVID-19 less reliable.
Laboratory IMAs include enzyme immunoassay (EIA) and
● prevailing community incidence of covid-19, together with diagnostic enzyme-linked immunosorbent assay (ELISA), radioimmunoas­
test accuracy, must be considered in the management of all sus­ say (RIA), fluoroimmunoassay (FIA), chemiluminescent immu­
pected covid-19 cases
noassay (CLIA) and counting immunoassay (CIA), and all of
them are affected by: 1) technical reasons in each type, 2)
testing in window period, and 3) antibody-related parameters.
Generally, IMAs are affected by specific endogenous antibo­
dies (heterophile, autoantibodies, antinuclear or anti-animal)
of accuracy, rapid turnaround times, and which can be imple­ or exogenous administered antibodies (Ig-drugs) that interfere
mented by inexperienced laboratory staff, have become and give a falsely elevated result in one assay or a lower result
widely available in clinical practice. Most RDTs are designed in another assay, even in the same individual [25].
on the basis of lateral-flow immunoassay (LFIA), and they are False test results are present in each IMA type, interpreta­
currently used for a qualitative and to some extent quantita­ tion of serological tests’ sensitivity varies [26], and false-
tive COVID-19 monitoring in public or private non-laboratory positive serology test results have been reported in COVID-
environments. Sajid et al. [7] present Ag-RDTs (antigen-RDTs) 19 [27,28]. Serological assays show a various sensitivity range
as devices consisting of prefabricated strips of a carrier mate­ [29,30].
rial with dry reagents, activated when applying the recom­ Real-time PCR is the technique of collecting data through­
mended specimen. Similar assays, but which rapidly detect out the PCR process as it occurs, and rPCR can amplify DNA,
antibodies targeting SARS-CoV-2, include Ig-RDTs (immuno­ or, when preceded by a reverse transcription, RNA. The thresh­
globulin-RDTs). old cycle (Ct) is the point of time at which the target amplifi­
Five type of false-positive Ag-RDT interpretations are recog­ cation is first detected, and fluorescence intensity is greater
nized: 1) errors in test operation, 2) poorly specific Ag-RDTs, 3) than background fluorescence [31,32]. Viral load is inversely
detection of inactive or residual SARS-CoV-2, 4) cross- related to the Ct value, with lower Ct values correlating to
contamination and 5) cross-reactions with other substances higher viral density in clinical specimens. Yet, it is not deter­
in clinical samples. mined as varies among diagnostic technologies and fluores­
False Ag-RDT results arise when test procedures are incorrectly cence systems, and several manufacturers have launched
followed (improper sample handling, contamination of test kits or different RT-PCRs [33,34]. Nowadays, several rapid PCR assays
clinical specimens, deviation from flow through the sequence of are utilized, even combining lateral flow technologies, or
test performance, lack of test validation) or by untutored users. Ag- named as closed PCRs (classical hand-performed are opened
RDT positivity does not exclude other infection, or co-infection with PCRs) that are known to be ‘RNA-RDTs’. Yet, several rapid
coronaviruses other than SARS-CoV-2, as many test kits are assays lack in control existence and, thus, test validity is
designed to detect highly conserved proteins [8,9]. Highly sensitive risky; additionally, they are affected by bloody and viscous
tests may detect inactive virus, or virus at low density in clinical samples.
specimens. Endogenous (e.g. blood) or exogenous (e.g. nasal spray In routine laboratory PCR testing, some false-positive
ions, or chemicals that affect the pH of the test cassette) may results can be managed through standard curve or interim
impact on test performance, giving rise to false-positive results controls [35]. However, misleading results can occur due to: 1)
[10]. LFIAs may be susceptible to temperature fluctuations, humid­ inadequate laboratory rRT-PCR experience, 2) SARS-CoV-2
ity, and positioning of the cassette during the testing procedure cross-contaminations, 3) detection of unspecified corona­
[11,12]. viruses, 4) SARS-CoV-2 inactive/residual detections, 5) cross-
Tzouvelekis et al. [13] reported the first false-positive Ig-RDT case reaction with nucleic acids from other pathogens or tissue
in July 2020; yet, other authors have announced cross-reactivities cells, and 6) technical reasons relating to kit primers, probes
with other viruses [14,15]. Heretofore, false-positive Ig-RDTs are and fluorescence type.
present due to:1) an erroneous Ig-RDT operation, 2) use of poorly Generally, cross-contaminations in laboratories, especially
specific Ig-RDT assays, 3) inattention to the time constraints in two-step rRT-PCR (processing RNA extraction and polymer­
imposed during a single test, and 4) cross-reactions with other ization in different tubes), while sampling or handling, are
sample substances. possible [2,36]. Temperature is crucial for whole PCR proce­
The use of Ig-RDTs during the resolution of SARS-CoV-2 infection dure. Van Kasteren et al report that some assays detect both
may be misleading, as there is uncertainty as to the duration of SARS-CoV-2 and SARS-CoV, because targeted genetic regions
persistence of IgG following primary and recurrent SARS-CoV-2 share homology [37], other pathogens, respiratory tract or
infection [16,17]. Endogenous factors, such as hematocrit levels or colon organisms. Lan et al report positive RT-PCR tests in
other blood substances, can affect the whole LFIA procedure in cases who have recovered from COVID-19 [38], but the assay
 EXPERT REVIEW OF RESPIRATORY MEDICINE 3

cannot distinguish between viable virus and noninfectious or the positivity, but in the first case, the extra antibodies inter­
residual RNA, whereas viral shedding is related to infectivity fere by separating and binding to the control and the target­
[39]. Regarding fluorescence, prime-dimers (detected in classi­ ing antibodies, thus blocking the reaction.
cal RT-qPCR via melting curve), short oligonucleotide primers Approaching the ‘gold-standard’ rRT-PCR and the extrac­
and probes, or fluorescent dyes that bind nonspecifically to tion-free technologies, some common false-negative types
dsDNA even to ssDNA, can give rise to false-positive results, occur in: 1) inadequate laboratory rRT-PCR performance, 2)
while various methods use different genes and different sample deficiency or degradation, 3) technical reasons relating
probes that may not be equivalent, and, thus, there is a 100- to kit primers, probes and fluorescence type, 4) SARS-CoV-2
fold difference in limit of detection (LoD) between some mutations and 5) RT-PCR inhibitors. Faulty sample collection,
assays [40,41]. processing, transportation, or degradation of the SARS-CoV-2
RNA during shipping/storage, can lead to suboptimal rRT-PCR
test performance and false-negative SARS-CoV-2 results.
2.2. False-negative test results
Viral load and Ct affect result accuracy, while applying
As previously stated, a negative result does not rule out the a cutoff could reduce false-positive and increase false-
presence of SARS-CoV-2 infection. Common causes of false- negative test results [30]. However, in some tests, false-
negative Ag-RDT tests include: 1) faulty technique in operating negative results occurring through lack of cell material in the
the assay, 2) insufficient clinical specimens, 3) inhibitors, and 4) sample are controlled for by simultaneous detection of
antigen degradation. a universally expressed human gene. Most tests present
LFIA's performance depends on numerous factors, while lumi­ a LoD for the number of viral copies that can be detected,
nescent and fluorescent LFIAs have higher sensitivity [42]. and false-negative tests may arise if the viral load is lower than
Inexperienced operators who may be deployed to run large that detection limit [2]. Poor sample quality or collection in
volumes of Ag-RDTs for COVID-19 monitoring, may handle assay very early or late infection could give rise to a false-negative
materials poorly or interpret tests incorrectly, compounding the test, depending on the assay’s sensitivity [2]. Also, sample
rates of false-negative tests. Each tests’ result is affected by the degradation is a possible etiology for a false-negative PCR
dynamics of SARS-CoV-2 viral load in early pre-symptomatic and test result. Furthermore, SARS-CoV-2 may not be detected, or
later stages of viral shedding [43]. An insufficient sample or viral may give rise to ambiguous test results, if the genome expres­
mutation can cause a false result, and an individual may have the sing target genes is mutated [2]. The fluorescent system,
virus, but the swab might have not collected it from nose or throat. which plays a crucial role in the final result, may be affected
Apart from possible exogenous substances, endogenous molecules by PCR inhibitors in the sample. Also, pooling strategies, as
could clog the membrane at the cassettes’ conjugate pad in high laboratory methods in PCR assays can be risky for giving rise
concentrations. Certain ‘sandwich’ LFIAs may give rise to false- to false-negative test results. A brief synopsis of the etiologies
negative results when samples are saturated with antigen: the so- for false test results is illustrated in Table 1.
called Hook effect [44].
Ig-RDTs can test negative in the presence of SARS-CoV-2
3. Respiratory prevention
infection due to: 1) erroneous operation of the assay, 2) factors
which may impair antibody production, 3) insufficient sam­ NAAT are currently the ‘gold standard’ assays for the detection of
ples, 4) inhibitors, and 5) antibody degradation. SARS-CoV-2 in clinical specimens. Because of suboptimal test sen­
Palma et al. [45], Childs et al. [46], Taneja [47] and other sitivity and specificity, false-negative and false-positive results may
authors report several factors that impact on antibody produc­ occur. False-negative results, which lead to a failure of detecting
tion, such as sex, diet, genetics, adjuvants, vaccines and other persons who are infected with SARS-CoV-2, are potentially more
parameters affecting immunity, and, thus, rapid or laboratory damaging than false-positive tests. It is essential that such false-
IMA's results are comparably affected. Moreover, handling and negative test results be minimized, so that respiratory physicians
sampling that lead to antibody degradation can give rise to and other clinical staff caring for such patients are alerted to the
false-negative results. Autoimmune conditions and treatment correct diagnosis as soon as possible, especially when hospitaliza­
thereof may also give rise to false-negative tests for SARS-CoV tion and further treatment strategies are necessary. The PCR assay
-2 antibody [48,49]. Deeks et al. [3] report that most cases of on respiratory specimens may be inhibited in several ways, apart
symptomatic SARS-CoV-2 infection will test positive for anti­ from a SARS-CoV-2 mutant that cannot be detected by the assay,
bodies directed against the virus. When a patient is early ill, and respiratory physicians should be trained to preempt false-
IgM/IgA antibodies may not be peripherally detectable, and negative test results, in COVID-19 or other pathogens requiring
IgA, IgM and IgG antibodies present a sensitivity heterogene­ a PCR assay identification.
ity [3]. Also, endogenous and exogenous factors can affect the PCR inhibitors act on one or more essential stages of the PCR
final result. Hematocrit, triglycerides, cholesterol (as the cellu­ testing procedure, from nucleic acid binding, capture or degrada­
lose-based material into the cassette LFIA is hydrophilic and tion, DNA polymerase inhibition, or ionic buffer alteration which
affected by viscosity), hemoglobin, and sample temperature, may increase the Ct value and give rise to false-negative test results.
could affect the final result in some cases [18,19]. In traditional When referring to RT-PCR, reverse transcriptase can be inhibited,
lateral flow serodiagnostic formats, the degree of detectable too. Schrader et al. [50], Wilson et al. [51] and Sidstedt et al. [52,53]
binding is reduced in the presence of high concentrations of present several substances (including hemoglobin, lactoferrin, mel­
nonspecific immunoglobulin. Laboratory IMAs’ negativity is anin, IgG, myoglobin, NaCl, tannic acids, urea, bile salts, bilirubin,
being affected by antibody interference at the same way as cellulose, heparin, free radicals and ethanol) which, when present in
4 D. S. MOULIOU AND K. I. GOURGOULIANIS

Table 1. Synopsis of false COVID-19 test results potential reasons in all test types. Each test varies in specificity and sensitivity, and a positive test does not exclude
the presence of another pathogen or co-infection. SARS-CoV-2 vaccination does not exclude other pathogen or co-infection.
Potential reasons for COVID-19 false test results
erroneous test administration – untutored use – deviation from testing protocol
False-positive test result False-negative test result
Antigen Antibody Antigen Antibody
non-clear place – sampling/handling contaminations time of implementation – poor sampling – humidity – position – sample viscosity – temperature – time
humidity – position – sample viscosity – temperature to evaluation (early or late reading of the test result) – destroyed cassette –
sample degradation – time of evaluation – mutations
cross-reactions with other antigens cross-reactions with other antibodies Hook effect antibody production (e.g. age, sex,
diet, smoking, adjuvants, vaccines,
genetics,etc)
SARS-COV detection SARS-COV-2 vaccination SARS-CoV-2 inadequacy exogenous/endogenous other
antibodies
inactivate virus detection IgG positive long after initial infection late test implementation (long after early test implementation (pre-
infection) symptomatic or asymptomatic
cases)
exogenous factors (e.g. high exogenous factors (e.g. high exogenous factors (e.g. high exogenous factors (e.g. Ig-drugs, etc)
concentrations of nasal spray, concentrations of nasal spray, concentrations of nasal spray,
chemical substances or ions) chemical substances or ions) chemical substances or ions)
endogenous factors (e.g. blood- endogenous factors (e.g. hematocrit, endogenous factors (e.g. blood- endogenous factors (e.g. hematocrit,
impurity derived substances) etc) impurity derived substances) etc)
nucleic acid amplification test (RT-PCR) nucleic acid amplification test (RT-PCR)
nonclear place – sampling/handling contaminations – temperature deficient sampling – suboptimal processing/RNA extraction – temperature
technical reasons (e.g. prime-dimers, short/nonspecific primers, probes, technical reasons (e.g. destroyed reagents – nonspecific primers, probes,
fluorescence) fluorescence)
Ct cutoff value/control in different test interim guidances Ct cutoff value/control in different test interim guidances
cross-contaminations in sampling, handling, laboratory (especially in 2-step RT- PCR inhibitors
PCR)
cross-reactions with other pathogens/tissue nucleic acids or SARS-COV detection SARS-COV-2 nucleic acid degradation
inactive/residual SARS-COV-2 detection SARS-COV-2 genome mutations

high concentrations, have been found to affect test performance. and there exists a report of a COVID-19 case with a false-
Combs et al. [54] and Kuffel et al. [55] report metal ions that affect negative PCR assay, presenting a past medical history of
PCR performance, and Marino-Merlo et al. [56] show that reverse alcohol use disorder, but it remains unknown if alcohol
transcriptase can be inhibited from antiretroviral drugs. The capa­ was consumed before sampling [50,57]. Important exogen­
city of each PCR test kit to perform in the presence of different ous factors that affect PCR assays are specific drugs that
inhibitors have been presented in interim guidance documents. may exist in respiratory tract specimens, such as nasal
Some PCR kits have controls to detect inhibitors, while others sprays including humic and fulvic acids’ derivatives, pheno­
cannot detect these substances. lic ions, polysaccharides, polyamines, etc., that mainly inhi­
Respiratory tract samples include tissue residues and respiratory bit DNA polymerization. For instance, inhaled drugs with
secretions, with endogenous and exogenous factors, initially depos­ specific chemical substances, such as inhaled heparin for
ited in the respiratory mucosa or lung parenchyma which, in high pulmonary function, or inhaled D-cycloserine, may affect
concentrations, can inhibit inexpertly conducted or low-sensitivity the final result, while a high concentration of intranasal
PCR tests. Lower respiratory tract specimens are particularly prone cellulose powder may lead to nucleic acid or polymerization
to being affected by different pulmonary pathologies which lead to inhibition. Furthermore, chemotherapy drugs, such as bleo­
variability of specimen adequacy (bloody, viscous, etc), while upper mycin, may affect PCR, as the inhibition mechanism is
respiratory tract specimens tend to be affected by exogenous almost the same, and especially bleomycin can give rise to
factors such as drugs and inhaled toxins. bleomycin-induced pneumonitis (BIP) which provides
Generally, occupational and allergic lung diseases, such as occu­ a saturated bronchoalveolar lavage (BAL) specimen for
pational asthma or chronic obstructive lung disease (COPD) with an a low sensitive PCR assay. Last but not least, antiretroviral
ongoing exposure, could affect respiratory sample testing by contain­ therapy could inhibit RT-PCR assay, since some antiretroviral
ing specific known inhibitors. Occupational and interstitial lung dis­ drugs are even being tested for their effectiveness in
eases (ILD), such as asbestosis, silicosis, alveolar microlithiasis, reverse transcriptase inhibition assays.
chemical pneumonia, melanoma or hemorrhage-related diseases, As it appears, cases with preexisting conditions that could yield
could affect a PCR test by presenting sample inhibitors (metal ions false-negative test results, should be reported from physicians to
or blood substances). Respiratory conditions (endogenous factors) laboratory experts. Alternative and more sensitive assays can be
that may impact on the performance of RT-PCR are presented in performed in laboratories -than the classical methods for non-
Figure 1. bloody or non-viscous samples-, or combination of PCR assay and
PCR assay is affected by ethanol, contained in a sample, IMAs, so as to prevent PCR inhibition. Thus, a potential false-
thus consuming alcohol just before sampling could be risky, negative COVID-19 case can be prevented.
 EXPERT REVIEW OF RESPIRATORY MEDICINE 5

Figure 1. Respiratory prediction of an endogenous inhibited PCR result.

High concentrations of endogenous or exogenous substances, if present in upper or lower respiratory tract samples, can lead to false-negative PCR results. These inhibitory factors apply to
PCR testing for all respiratory pathogens tested in PCR assay, and are not limited to SARS-CoV-2 testing.

4. Preexisting conditions and testing assays result can occur again, and arise suspicions for a SARS-CoV
-2 infection, especially when the total health status is
As it appears, not only the clinical diagnosis but also the laboratory
ambiguous. As mentioned before, anti-SARS-CoV-2 antibo­
test result interpretation is affected by a case’s preexisting diseases
dies can interfere in other IMAs.
and total health status. In general, false-positive COVID-19 cases are
PCR assays can be affected by preexisting medical con­
more likely to exist in a low COVID-19 prevalence in the community
ditions, such as jaundice and hyperbilirubinemia-related dis­
where other viruses abound, and false-negative COVID-19 are more
orders, since high concentrations of bilirubin and bile salts
likely to exist in a community rating other comorbidities. Since
found in human samples can inhibit PCR [50].
COVID-19 Ag-RDTs were initially based on SARS-CoV highly con­
Concomitantly, PCR assay is affected by the material of the
served genetic loci, it is certain that they can present a false-positive
sample collector (swab, etc). Background medical conditions
result for SARS-CoV-2 [8], and less accurate Ag-RDTs show
leading to an excess of specific proteins (collagen, ferritin,
a positivity to influenzas, other viruses, or bacteria. If sample con­
lactoferrin, myoglobin, IgG, hemoglobin and heme) in
tains blood, there is a possibility for blood-derived substances and
human samples, can be crucial in estimating a PCR test
antibodies to interfere in the assay, as occurring in the IMAs.
result [50–53]. Phenolic, citrates, polyamines or polysacchar­
Rapid (Ig-RDTs) and laboratory IMAs (such as ELISA) are more
ides found in human samples, due to preexisting conditions
likely to be affected by the preexisted individual’s immunity, such as
or because of specific drugs’ consumption and metabolism,
several autoimmune diseases [13,22]. Also, Ig-RDTs have indicated
need to be further taken into account, in accordance with
a false-positivity even in pregnant women [58]. IMAs’ interference
the interim guidances of each implemented assay.
has been reported in specific diseases producing heterophile anti­
Alternatively, these cases with preexisting conditions that
bodies, such as infectious mononucleosis (IM) [59]. More than a half
could affect a testing assay performance should be reported
of the patients’ samples contain HAAAs [25,60] that interfere in
from physicians to laboratory experts. Better and more sen­
IMAs by presenting both positivity and negativity, while existing
sitive assays can be utilized instead of, and may some false
mainly in serum of animal workers, people living with indoor pets
results be prevented and eliminated.
or patients being administered genetically engineered mouse
monoclonal antibodies (Ig-drugs) for therapy or imaging. In reality,
all human beings present autoantibodies interfering in IMAs and,
5. Management strategies
thus, every serological test should be interpreted according to an
individual’s total health condition [61,62]. Rheumatoid factor (RF) The WHO recommends that challenging cases, so-called chal­
and antinuclear antibodies have long been reported for serological lenging COVID-like diseases (CLD), be tested for other respira­
interferences [63–65]. HIV, hepatitis, syphilis, malaria, lupus, vascu­ tory pathogens, as co-infection with other respiratory
litis, hyper-gamma-globulinaemia and presence of HLA-DR antibo­ pathogens is frequent. Parallel testing platforms are becoming
dies have long been correlated with false IMAs test results and more widely available [70,71]. SARS-CoV-2 is now ubiquitous,
antibody interference [66–69]. and any suspected case should be tested for SARS-CoV-2
Importantly, while trying to detect the real infection with regardless of whether another respiratory pathogen is
another disease’s IMA, instead of targeting COVID-19, a false detected [2].
6 D. S. MOULIOU AND K. I. GOURGOULIANIS

Management decisions rely on NAAT, combined with med­ to detect the specific tests’ molecular reason of a false result
ical examination, epidemiological information, and patient and prevent further misleading results.
history, and the results of the diagnostic work-up inclusive of
all radiological, biochemical and microbiological tests. Espy
et al. [35] consider IMAs as an essential tool for the diagnosis 6. SARS-CoV-2 vaccination
of viral infections. During the course of the pandemic, SARS-
CoV-2 should be considered in all acutely ill patients present­ In the era of growing access to SARS-CoV-2 vaccination, ser­
ing with respiratory failure, and the virus, or its mutants, may ologic testing to establish a diagnosis of SARS-CoV-2 infection
or may not be present in the patient’s respiratory secretions. will become more complicated. Numerous vaccine platforms,
A combined assessment is critical for the rational manage­ ranging from mRNA vaccines, antigen-based vaccines and viral
ment of such patients. An example of such a COVID-19 man­ vector vaccines, are currently under investigation or have
agement algorithm is presented in Figure 2. been implemented in mass immunization campaigns. Ag-
False-positive COVID-19 place patients at risk through RDTs do not detect SARS-CoV-2 antigen derived from antigen-
cohorting with other COVID-19 cases, while false-negative based vaccines; nevertheless, it remains unknown as to
COVID-19 place healthcare workers, other patients and the whether tissue or blood impurities in respiratory specimens
general public at risk for infection from an undiagnosed could give rise to false-positive Ag-RDT test results.
source case. Both scenarios have the potential to impact sub­ Ig-RDTs cannot be relied upon to establish a diagnosis of
stantially on patient-level care and public safety. A positive SARS-CoV-2 infection in individuals that have received SARS-
test does not exclude co-infection with other respiratory CoV-2 vaccines. It remains to be seen how vaccine-induced
pathogens, while a negative test does not exclude SARS-CoV SARS-CoV-2 antibody responses may cross-react with serologic
-2 infection, particularly in the context of infection with viral tests for other pathogens, giving rise to false-positive test
mutants. Where the clinical index of suspicion is high, results for those pathogens. SARS-CoV-2 vaccination history,
repeated testing should be undertaken. and timing thereof, should be established when consideration
Despite the false COVID-19 tested cases, it is clear that is being made to use Ig-RDTs to screen for current or past
a result represents a unique tested sample in a particular SARS-CoV-2 infection. It would be appropriate to use Ig-RDTs
point of time; therefore, the whole case condition can be that target different antigen-antibody loci from the vaccine
different, and, exclusively, each cases’ various samples can antigen when using Ig-RDTs in persons who have received
manifest different results. Comparing different clinical cases SARS-CoV-2 vaccination.
with false results, in different test types, methods and kits, Considering that various platforms are being under consid­
when even an individuals’ samples vary in the same test kit, eration SARS-CoV-2 vaccination, including multi-epitope or
seems groundless. Instead of randomly reporting and analyz­ reverse vaccinology and immunoinformatics [72–75], may
ing clinical cases with false results in numerous test types, these technologies be more precise in immunogenic
precluding each tests’ limitations, it would be more successful responses. Also, may multi-allelic vaccines be more

Figure 2.. Algorithm for the management of suspected COVID-19 cases during the course of the pandemic.
Ag-RDT: Antigen rapid diagnostic test, Ig-RDT: Immunoglubin rapid diagnostic test. Color represents symptomatic (sympt), suspected (susp), contacted (cont), and non-suspected (non-susp)
cases. Symptomatic and suspected cases, as well as cases with possible/confirmed contacts, or cases with no history, and evidence for infection with SARS-CoV-2, is shown here. As no test
is 100% accurate and false results can occur, COVID-19 positivity is stated as ‘current evidence’ with precautions. Both false-positive and false-negative test result possibility is depicted in
each test type. Regarding Ig-RDTs, positivity for IgM/IgA and/or IgG should be combined with all the aforementioned criteria. CLDs should be considered in every testing type and further
management. RDTs are not to be existed necessarily, the algorithm is simply summarizing the whole testing cases.
 EXPERT REVIEW OF RESPIRATORY MEDICINE 7

advantageous in non-interfering in IMAs targeting the real tests’ performed by non-guidanced arise awareness about the
antibodies, as the natural antibody is different. However, in sampling quality and adequacy, the if-swab safe usage, and
this case, if a vaccine induces multi-epitope immune the final assessment regarding background status.Easier auto­
responses, it gives rise to additional produced antibodies in mated methods may be designed, such as ‘licking-devices’,
the body and, as a result, the possibility for a general IMAs’ since a virus exists in droplets and aerosols.Public should be
possible interference is equivalently increasing. informed about the importance of a test’s interim guidances.
NAATs done on respiratory samples cannot detect vaccine- Also, more sensitive rapid LFIAs, different for winter/summer
derived SARS-CoV-2 nucleic acids which were administered via use – regarding humidity/temperature conditions, could be
the intramuscular route. It remains unknown as to whether designed.
blood or tissue contamination of respiratory fluids could give New technologies have loss of standardization as the
rise to false-positive SARS-CoV-2 NAATs in persons that have countless PCR kits vary in methods and cutoff values, thus,
been administered nucleic acid-containing SARS-CoV-2 vac­ test results are paralleled in unassociated weights, and
cines, however. a realistic comparison between cases is trammeled. Thus, by
preserving the existence of misleading COVID-19 cases in such
way, scientific community is being prevented from clear-
7. Conclusion
sighted advances. Since PCR assay cannot distinguish between
This review article has summarized what is currently known active and residual RNA, a better assay – maybe with an active
about false-negative and false-positive tests for SARS-CoV-2, viral amplicon size cutoff value- needs to be designed. Also,
and clinical scenarios that may give rise to such erroneous a further development of the widely successful CRISPR-Cas9
results. All tests should be interpreted with caution, within the method for a better detection and differentiation of amplifica­
context of the individual patient’s clinical status, exposure tion products could be seen in near future. The false-positive
history, and the results of ancillary tests, as well as in the COVID-19 test results in other existing pathogens need further
context of the prevalence of SARS-CoV-2 infection in the analysis. Besides, it remains unknown, to what extent, in cases
wider community at the time of testing. In situations where with a negative NAAT and positive IMA, the final result could
SARS-CoV-2 is circulating widely, the positive predictive value be a negative COVID-19 case, as antibodies are such difficult
of the available tests increases. Where clinical suspicion is to be assessed. Also, the real time that SARS-CoV-2 is detected
high, despite an initial negative test of SARS-CoV-2, tests active and inactive in deceased cases is unclear.
should be repeated in order to establish a firm diagnosis of SARS-CoV-2 vaccination era boosted the mRNA technology
the condition. and lipid nanoparticles with mainly the intramuscular way;
It seems obvious that not only is a microcosmic high- therefore, gene markers, such as GFP (green fluorescent pro­
affinity reaction crucial for confirming a realistic COVID-19 tein) could be more useful, in detecting exactly in which tissue
case, but also clinicians and respiratory physicians should be cells apart from muscle cells can these vaccine nanoparticles
in a great macrocosmic interaction, for an accurate test result, exist, for an accurate prognosis and exclusion of a possible
further respiratory medical management and treatment per­ false-positive result. Also, it would be essential for determining
spectives. COVID-19 can be such an illusory disease; yet, chem­ further vaccine side-effects when analyzing exactly the nano­
istry cannot be deceived, and some false result cases can be particles’ tissue route. Furthermore, the in vivo vaccine-
predicted. produced anti-SARS-CoV-2 antibodies should be analyzed for
Relentless pursuit of SARS-CoV-2 in a patient who has possible cross-reactions in other pathogens’ serology assays –
multiple negative tests, despite using different assay types a possible false-positive test result in other pathogen.
and test kits, should not be encouraged, however. The kinetics of pulmonary route need to be further studied
Management strategies can be precise and straightaway, for a future vaccine or therapy progression, against COVID-19 or
when assessing each test result at the angle of each testing other lung infections, even for preventing the so-called coming
method guidelines – concerning vaccination-, and, respiratory ‘Disease X’ severity. Preclinical models and researches for
physicians can prevent some potential false tested cases for inhaled antibodies or vaccines need to speed up, for lung-
a better and on-the-spot response to emergent COVID-19 targeted viral drugs or pulmonary-based vaccinations.
cases. Inhalation-based or intravenous strategies targeting solely the
lungs or lung-designed vaccines/drugs with lung-cell signal
peptides, are more successful. They may need lower doses
8. Expert opinion
reducing chances for exposing toxic side-effects in other tissues.
After almost a year of SARS-CoV-2 pandemic, the various Targeting the primordial system of the lung tissue-resident
reports of falsely tested cases, precluding each tests’ limita­ innate immunity, could be a more promising strategy for SARS-
tions, have revealed that physicians are far away from the real CoV-2 or other current or future lung infections’ drug or vaccine
tests’ capacity, so their sole point-of-care performing, could be formulation. Lung dendritic cells (DCs) named the lung sentinels,
more effective in estimating a test result. In the decades of have proved to be important in the initiation of antiviral
multiplexed and rapid NAATs, rapid LFIAs, and golden nano­ responses that lead to general viral clearance, so they could be
particles which present no research endpoint, the testing a future potential vaccines’ antigen expression target. However,
automation, provides better accuracy and sample handling, lung immunity and DCs need to be thoroughly analyzed, as
shorter turnarounds and cost-effective administrations for there are several functional DCs’ questions in common respira­
the improvement of a pathogens’ detection. However, ‘self- tory diseases, such as COPD, to prevent possible side effects.
8 D. S. MOULIOU AND K. I. GOURGOULIANIS

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employment, consultancies, honoraria, stock ownership or options, expert
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